Method for developing organ that lacks specific functional cell

ABSTRACT

Provided is a method for developing a secondary organ by using a non-human animal in which organ formation is inhibited, for the purpose of establishing a process for producing a functional cell such as a β cell within the body of an animal such as a pig, the method including the step of raising a newborn or a fetus of the non-human animal in which organ formation is inhibited by complementing at least a part of the function of the organ whose formation is inhibited.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of copending application Ser. No.16/638,440, filed on Feb. 11, 2020, which is the National Phase under 35U.S.C. § 371 of International Application No. PCT/JP2018/034390, filedon Sep. 18, 2018, which claims the benefit under 35 U.S.C. § 119(a) toPatent Application No. 2017-178552, filed in Japan on Sep. 19, 2017, allof which are hereby expressly incorporated by reference into the presentapplication.

TECHNICAL FIELD

The present invention relates to a method for developing a secondaryorgan by using a non-human animal in which organ formation is inhibited.The present invention also relates to a non-human animal that has asecondary organ lacking a specific functional cell. Furthermore, thepresent invention relates to a method for producing a cell. Furthermore,the present invention relates to a secondary organ lacking a specificfunctional cell.

BACKGROUND ART

Blastocyst complementation method is a method for forming an organderived from cells of a normal animal by injecting the cells of thenormal animal into a blastocyst of an animal lacking an organ. Utilizingthis blastocyst complementation method, the present inventors havesuccessfully generated a chimeric pig that had a pancreas derived from adonor by injecting an embryo from a normal pig (donor embryo) into anembryo from a pig lacking a pancreas (host embryo) and allowing the hostembryo to develop in the body of a surrogate parental pig (Non PatentLiterature 1). Although the chimera was generated between pigs in NonPatent Literature 1, a pig that has a human pancreas is expected to begenerated by using a human pluripotent stem cell as a donor and such ahuman pancreas may be used for islet transplantation.

CITATION LIST Non Patent Literature

-   Non Patent Literature 1: Matsunari et al., PNAS 110: 4557-4562    (2013)

SUMMARY OF INVENTION Technical Problem

If a human organ (for example, a pancreas) or a functional cell (forexample, a β cell) can be successfully produced within the body of ananimal such as a pig, the problem of donor shortage in transplantationtherapy can be solved.

On the other hand, under the existing circumstances, there are manyethical problems to be solved in transplantation in which a chimericstate between a human and an animal is induced at an early stage ofdevelopment and an organ or functional cell thus produced istransplanted into a human. In comparison with this, it is believed thatthere is a lower ethical barrier to performing cell transplantation inwhich a chimeric state between a human and an animal is not induced atan early stage of development, a specific functional cell (for example,a β cell) that can establish itself in a human is grown in an organ ofan animal such as a pig, and the functional cell is transplanted into ahuman.

However, until now, there has been no known organ or tissue suitable forcell growth to perform such cell transplantation, wherein the organ orthe tissue lacks only the specific functional cell (for example, a βcell) and serves as an environment for producing and growing the abovedescribed specific functional cell.

Under such circumstances, it is an object of the present invention toprovide means for producing a functional cell within an animal body.

Solution to Problem

A newborn piglet in which pancreas formation is inhibited dies aboutthree days after birth due to hyperglycemia and dyspepsia. The presentinventors raised such a newborn by administering insulin and a digestiveenzyme combination thereto so that the newborn survived, and have foundthat as the newborn grew, pancreas-like tissue formed in a space where apancreas would have formed under normal circumstances. The presentinventors also have found that this pancreas-like tissue came to producea digestive enzyme and glucagon but did not come to produce insulin (inother words, no β cell formed). It was totally unexpected that thispancreas-like tissue that had not been found during the fetal stagestarted to develop by the above described raising and further formedinto an incomplete pancreas in which no insulin-producing β cell formedwhile other endocrine cells and/or an exocrine cell existed. It was alsototally unexpected that only insulin was not produced although thedigestive enzyme and glucagon were produced.

The β cell-lacking pancreas-like tissue formed by the above describedraising has an environment (such as a tissue structure and function)other than the β cell and is believed to be an excellent “vacant niche”for growing a progenitor cell of the β cell and a pluripotent stem cell.Therefore, it is believed that a human β cell can be producedefficiently by transplanting, for example, a progenitor cell of a β cellderived from a human into the β cell-lacking pancreas-like tissue.

The present invention has been accomplished on the basis of the abovedescribed findings.

Specifically, the present invention provides the following [1] to [18].

-   [1] A method for developing a secondary organ by using a non-human    animal in which organ formation is inhibited, including the step of:

(1) raising a newborn or a fetus of the non-human animal in which organformation is inhibited by complementing at least a part of the functionof the organ whose formation is inhibited.

-   [2] The method according to [1], wherein the secondary organ    develops in such a manner that at least a part of the function    complemented by the raising is impaired.-   [3] The method according to [1] or [2], wherein the secondary organ    is an organ lacking a specific functional cell.-   [4] The method according to [3], wherein the functional cell is a β    cell.-   [5] The method according to any of [1] to [4], wherein organ    formation is inhibited by genetic modification of the non-human    animal in step (1).-   [6] The method according to any of [1] to [5], wherein the organ    whose formation is inhibited is a pancreas and complementation of    the function of the pancreas whose formation is inhibited is    performed by administering at least insulin and a digestive enzyme.-   [7] The method according to [5], wherein the genetic modification is    overexpressing a Hes1 gene under the control of a Pdx1 promoter in    the non-human animal.-   [8] The method according to any of [1] to [7], wherein the non-human    animal is a pig.-   [9] A non-human animal obtained by raising a newborn or a fetus of a    non-human animal in which organ formation is inhibited, wherein the    non-human animal has a secondary organ lacking a specific functional    cell.-   [10] The non-human animal according to [9], wherein the organ is a    pancreas and the functional cell is a β cell.-   [11] The non-human animal according to [10], wherein the newborn or    the fetus of the non-human animal is that in which organ formation    is inhibited by overexpressing a Hes1 gene under the control of a    Pdx1 promoter.-   [12] The non-human animal according to any of [9] to [11], wherein    the non-human animal is a pig.-   [13] A secondary organ developed by raising a newborn or a fetus of    a non-human animal in which organ formation is inhibited, wherein    the secondary organ lacks a specific functional cell.-   [14] The secondary organ according to [13], wherein the organ is a    pancreas and the functional cell is a β cell.-   [15] The secondary organ according to [14], wherein the newborn or    the fetus of the non-human animal is that in which organ formation    is inhibited by overexpressing a Hes1 gene under the control of a    Pdx1 promoter.-   [16] The secondary organ according to any of [13] to [15], wherein    the non-human animal is a pig.-   [17] The secondary organ according to any of [13] to [16], wherein a    transplanted cell established itself in the secondary organ.-   [18] A method for producing a cell within the body of a non-human    animal, including the step of raising the non-human animal according    to [1] or [2].

This specification encompasses the content described in thespecification and/or drawing of Japanese Patent Application No.2017-178552, which forms the basis of priority of this application.

Advantageous Effects of Invention

The present invention provides the method for developing a secondaryorgan by using a non-human animal in which organ formation is inhibited,the non-human animal that has a secondary organ lacking a specificfunctional cell, the method for producing a cell, and the secondaryorgan lacking a specific functional cell.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a photograph of internal organs of a pig in which pancreasformation is inhibited (right) and a wild type pig (left).

FIG. 2 shows a graph showing change over time in the weight (top) andthe blood glucose level (bottom) of the pig in which pancreas formationis inhibited and the wild type pig.

FIG. 3 shows a table showing blood biochemical parameters of the pig inwhich pancreas formation is inhibited and the wild type pig (23 days oldand 60 days old).

FIG. 4 shows a photograph showing the result of immunostaining of thepancreatic tissue of the pig in which pancreas formation is inhibited(right) and the wild type pig (left) (38 days old). The pancreas of thepig in which pancreas formation is inhibited has a pancreatic islet-likestructure including a glucagon-producing cell but production of insulinwas not observed.

FIG. 5 shows a photograph of a secondary pancreas of the pig in whichpancreas formation is inhibited (right) developed by supported raising(raising supported by administration of insulin and a digestive enzyme),and the pancreas of the wild type pig (left). It has an enough capacityfor injecting cells from the outside.

FIG. 6 shows a photograph showing HE staining of the pancreatic tissueof the pig in which pancreas formation is inhibited developed bysupported raising (lower row) and the pancreatic tissue of the wild typepig (upper row). The lobule of the pig in which pancreas formation isinhibited was smaller than the lobule of the wild type pig, but includedtissues other than β cells.

FIG. 7 shows a table showing the body weight, the pancreas weight, andthe ratio of pancreas weight to body weight of the pig in which pancreasformation is inhibited (upper row) and the wild type pig (lower row).

FIG. 8 shows a photograph showing the result of immunostaining foramylase of the pancreatic tissue of the pig in which pancreas formationis inhibited (right) and the wild type pig (left). An amylase-positivecell was observed in the pancreatic tissue of the pig in which pancreasformation is inhibited.

FIG. 9 shows a photograph of the pancreatic tissue of the pig in whichpancreas formation is inhibited implanted with a fibroblast. The leftphotograph is an HE staining image and the right photograph is afluorescent image. The transplanted fibroblast is stained with CM-DiI.

FIG. 10 shows an immunohistochemical staining image of the retina of thepig in which pancreas formation is inhibited (right) and the wild typepig (left). In the retina of the pig in which pancreas formation isinhibited, a neovessel has developed due to diabetic retinopathy.

FIG. 11 shows a photograph of the right eye of the pig in which pancreasformation is inhibited (right) and the wild type pig (left). The pig inwhich pancreas formation is inhibited has developed a cataractcharacteristic of diabetes.

DESCRIPTION OF EMBODIMENTS

Hereinafter, the present invention is described in detail.

(A) Method for Developing Secondary Organ by Using Non-human Animal inwhich Organ Formation is Inhibited

The method of the present invention for developing a secondary organ byusing a non-human animal in which organ formation is inhibited includesthe step of (1) raising a newborn or a fetus of the non-human animal inwhich organ formation is inhibited by complementing at least a part ofthe function of the organ whose formation is inhibited.

Examples of the organ whose formation is to be inhibited may include apancreas, and the organ may be any organ other than the pancreas as longas the newborn or the fetus can survive even if formation of the organis inhibited. Examples of the organ other than the pancreas may includea kidney.

Inhibition of organ formation can be performed by genetic modificationthat modifies a gene of the non-human animal having the organ. Forexample, inhibition of pancreas formation can be performed by using aPdx1-Hes1 gene composed of a Hes1 gene linked to a Pdx1 gene promoter,specifically by overexpressing the Hes1 gene under the control of thePdx1 promoter (Matsunari et al., PNAS 110: 4557-4562 (2013)). Organformation may also be inhibited by administration of an agent. On theother hand, the effect of the present invention cannot be achieved whenorgan formation is disabled by destroying the Pdx1 gene. Inhibition ofkidney formation can be performed, for example, by overexpressing aSix2-Notch2 gene (Fujimura et al., J Am Soc Nephrol, 21:803-810, 2010)and by inhibiting or suppressing expression of a gene that controlskidney development, such as Sall1 and Pax2.

The animal to be subjected to inhibition of organ formation may be anyanimal as long as the animal is not a human, but is preferably a mammal.Specific examples of the animal may include a mouse, a rat, a hamster, aguinea pig, a rabbit, a dog, a cat, a horse, a cow, a sheep, a pig, agoat, and a monkey. Among these, a pig can be a preferable animal.

In the present invention, a “secondary organ” means an organ-like tissuethat was formed in a site within the body of a non-human animal byraising and growing the animal, wherein the site is a site where aformation-inhibited organ (for example, a pancreas) would have formedunder normal circumstances. The secondary organ may have 100% or a partof the function of the normal organ. Examples of a preferable secondaryorgan may include a secondary organ lacking a specific functional cell.In this context, a “functional cell” means a cell that is included in anorgan and has some function, such as an a cell, a β cell, and a γ cellin the pancreas. Examples of a preferable functional cell may include aβ cell. Examples of a preferable organ lacking a specific functionalcell may include a pancreas lacking a β cell.

A newborn or a fetus of the non-human animal in which organ formation isinhibited can be obtained by a known method. For example, a newborn or afetus of a pig whose pancreas formation is inhibited by using thePdx1-Hes1 gene can be obtained by the following methods (a) to (d).

-   -   (a) Artificial insemination is performed on a wild type female        by using a sperm into which the Pdx1-Hes1 gene has been        integrated.    -   (b) In vitro fertilization is performed on an egg derived from a        wild type female by using a sperm into which the Pdx1-Hes1 gene        has been integrated.    -   (c) A male that produces a sperm into which the Pdx1-Hes1 gene        has been integrated is mated with a wild type female.    -   (d) A cloned pig is generated by nuclear transplantation using a        somatic cell into which the Pdx1-Hes1 gene has been integrated.

“Complementing at least a part of the function of the organ whoseformation is inhibited” means administering an enzyme or a hormone thatthe organ would produce under normal circumstances or an agent having afunction equivalent thereto; transplanting, for example, a cell thatsubstitutes for the function of the organ; using a device thatsubstitutes for the function of the organ; or the like. The function ofthe organ does not need to be fully complemented and may be partiallycomplemented as long as the newborn or the fetus of the non-human animalcan survive. A specific complementation method can be decided asappropriate depending on the organ type. For example, for complementingthe function of the pancreas, all or some of the hormones and digestiveenzymes produced by the pancreas may be administered. Specifically, thepancreas produces hormones such as glucagon, insulin, and somatostatin,all or some of which can be administered. The pancreas also producesdigestive enzymes such as a proteolytic enzyme (chymotrypsin andtrypsin), a polysaccharide-degrading enzyme (amylase), and a lipolyticenzyme (lipase), all or some of which can be administered. Furthermore,instead of administering the actual hormones and/or digestive enzymesproduced by the pancreas, an agent having a function equivalent theretomay be administered. Examples of a preferable raising method forcomplementing the function of the pancreas may include a raising methodthat includes administering insulin and the digestive enzyme. Examplesof the digestive enzyme to be administered in this context may include aproteolytic enzyme, a polysaccharide-degrading enzyme, and a lipolyticenzyme.

Raising the newborn or the fetus can be performed in accordance with anordinary method except that at least a part of the function of theformation-inhibited organ is complemented.

As described below, in some cases, human-derived cells are transplantedinto the secondary organ formed by the above described method, and suchheterotransplantation causes rejection. To avoid this rejection, it ispreferable to modify a gene of the non-human animal where the secondaryorgan is developed, thereby inducing immunodeficiency (SCID). Methodsfor inducing such immunodeficiency are reported in many publications,and the present inventors have generated an immunodeficient pig byknocking out an IL2RG gene (Watanabe et al., PLoS One 8(10): e76478(2013)). Thus, induction of immunodeficiency can be performed accordingto description of such publications. It is also possible to induceimmunological tolerance to a human-derived cell by transplanting a largequantity of human-derived cells into a fetus before immune system isestablished. Therefore, such a method may be used to avoid rejectioncaused by heterotransplantation.

The length of the raising period for developing the secondary organ isnot particularly limited. When the developed secondary organ is intendedto be used for a method described below for producing a functional cell,raising is continued until the functional cell is ready to be collected.

(B) Non-Human Animal

The non-human animal of the present invention is a non-human animalobtained by raising a newborn or a fetus of a non-human animal in whichorgan formation is inhibited, wherein the non-human animal has asecondary organ lacking a specific functional cell.

The non-human animal of the present invention can be used for the methoddescribed below for producing a functional cell, and furthermore canalso be used as a model animal for a disease or a malformation. Forexample, when the specific functional cell that the non-human animallacks is a β cell, which contributes to insulin production, thenon-human animal can be used as a model animal for a complication causedby diabetes, such as retinopathy, nephropathy, neuropathy, angiopathy,and cataract.

The type of the functional cell, the type of the secondary organ lackinga specific functional cell, and the type of the animal to be subjectedto inhibition of organ formation may be similar to those described inthe above “Method for Developing Secondary Organ by Using Non-humanAnimal in which Organ Formation is Inhibited.” The method for inhibitingorgan formation and the method for raising a newborn or a fetus can alsobe performed in a similar manner to the above “Method for DevelopingSecondary Organ by Using Non-human Animal in which Organ Formation isInhibited.”

As described above, in this specification, a “non-human animal,” whichis a “product,” is specified not by the structure or characteristicthereof but by a method for producing the same. The reason for this isthat the “non-human animal” is an organism and thus has an extremelycomplicated structure and characteristic, and therefore carrying outwork for specifying the structure and characteristic thereof requiresremarkably excessive economic expense and time.

(C) Method for Producing Functional Cell

The inventive method for producing a functional cell includes the stepsof (1) transplanting at least a progenitor cell of the functional cellor a pluripotent stem cell into the secondary organ of the non-humananimal; and (2) inducing the progenitor cell or the pluripotent stemcell to the functional cell.

A “progenitor cell of a functional cell” means a cell that originatesfrom a stem cell and can differentiate into a functional cell, andexamples thereof include a progenitor cell of an α cell, a progenitorcell of a β cell, and a progenitor cell of a γ cell in the pancreas.Examples of a preferable progenitor cell of a functional cell mayinclude the progenitor cell of a β cell. Instead of the progenitor cellof a functional cell, a pluripotent stem cell whose differentiation wasappropriately induced may be transplanted. An embryonic stem cell (EScell), an induced pluripotent stem cell (iPS cell), or the like can beused as the pluripotent stem cell. Other cells may also be transplantedin addition to the progenitor cell of a functional cell and thepluripotent stem cell. For example, in the case of the pancreas, a cellassociated with pancreatic islet formation is preferably transplanted.Examples of the cell associated with pancreatic islet formation mayinclude an interstitial cell, a cell involved in induction of a bloodvessel, and an endocrine cell.

The main purpose of the inventive method for producing a functional cellis producing a functional cell for transplantation into a human, andtherefore, the progenitor cell or pluripotent stem cell to betransplanted is preferably derived from a human.

The number of cells to be transplanted into the secondary organ can bedecided as appropriate depending on the type of the animal, the celltype, the type of the secondary organ, and the like. For example, whenthe cells are transplanted into the pancreas of a pig, the number ofcells is usually, without particular limitations, 1×10⁶ to 5×10⁸.

The timing for performing transplantation can be decided as appropriatedepending on the cell type, the type of the secondary organ, the type ofthe animal, and the like. When the cells are transplanted into thepancreas of a pig, transplantation is usually performed, withoutparticular limitations, at the timing when the pig is 10 to 90 days old.

When the cells to be transplanted are human-derived cells,heterotransplantation causes rejection. When the non-human animal intowhich cells are transplanted is the immunodeficient animal or the animalwith induced immunological tolerance to human-derived cells as describedabove, rejection can be avoided. Otherwise, it is preferable to avoidrejection by administering an immunosuppressive agent (for example,ciclosporin) to the non-human animal.

Although the secondary organ lacking a specific functional cell lacksthe specific functional cell, the secondary organ has other functionalcells and tissues, and thus, the secondary organ is presumed to have anenvironment favorable for differentiation into the specific functionalcell. Therefore, when progenitor cells or pluripotent stem cells aretransplanted into this organ, the cells are expected to differentiateinto the specific functional cells without requiring a particulartreatment. However, a substance such as an agent and a growth factor maybe injected into the secondary organ to promote differentiation into thespecific functional cell.

The functional cells are collected from the secondary organ at anappropriate time after transplantation. The timing for collection can bedecided as appropriate depending on the cell type, the type of thesecondary organ, the type of the animal, and the like. When β cells arecollected from the pancreas of a pig, the β cells are usually collected,without particular limitations, 1 to 12 months after transplantation.

Although a method other than the method of the present invention, forexample, a blastocyst complementation method can be used to produce ahuman-derived functional cell in a non-human animal, the inventivemethod is superior to a conventional method in the following respects.

(a) In the blastocyst complementation method, human-derived cells aretransplanted at an early stage of development such as a blastocyststage. Such transplantation at an early stage of development leads todilution of the human-derived cells. Specifically, some of thetransplanted human-derived cells differentiate into functional cells butthe majorities thereof differentiate into different cells other than thefunctional cells or disappear, which leads to a lower quantity ofresulting human-derived functional cells. In contrast to this method, inthe method of the present invention, human-derived cells aretransplanted into an already differentiated organ. Therefore, it isexpected that the majority of the transplanted cells differentiate intofunctional cells and abundant human-derived functional cells areobtained.

(b) In the blastocyst complementation method, human-derived cells aretransplanted into the blastocyst. The cells included in the blastocystare undifferentiated cells and these cells differentiate into adultcells over time. The transplanted human-derived cells cannot becomefunctional cells in the end unless the transplanted cells themselvesdifferentiate at the same pace as the differentiation progress of suchhost cells. Therefore, it is presumed that there are many human-derivedcells that cannot become functional cells because of relation to thedifferentiation progress of the host cells. In the method of the presentinvention, human-derived cells are transplanted into an alreadydifferentiated organ, and therefore, the above described problemassociated with the differentiation progress of the host cells ispresumed not to occur.

(D) Secondary Organ

The secondary organ of the present invention is a secondary organdeveloped by raising a newborn or a fetus of a non-human animal in whichorgan formation is inhibited, wherein the secondary organ lacks aspecific functional cell.

The type of the functional cell, the type of the secondary organ lackinga specific functional cell, and the type of the animal to be subjectedto inhibition of organ formation may be similar to those described inthe above “Method for Developing Secondary Organ by Using Non-humanAnimal in which Organ Formation is Inhibited.” The method for inhibitingorgan formation and the method for raising a newborn or a fetus can alsobe performed in a similar manner to the above “Method for DevelopingSecondary Organ by Using Non-human Animal in which Organ Formation isInhibited.”

EXAMPLES

Hereinbelow, the present invention will be described in more detail withreference to Examples, but the present invention is not limited to theseExamples.

Example 1 Obtaining Newborn Piglet in which Pancreas Formation isInhibited

The present inventors previously generated a chimeric pig that had apancreas derived from a donor by injecting an embryo from a normal pig(donor embryo) into an embryo from a Pdx1-Hes1 gene-carrying and pig inwhich pancreas formation is inhibited (host embryo) and then allowingthis to develop in the body of a surrogate parental pig (Matsunari etal., PNAS 110: 4557-4562 (2013)). A male of this chimeric pig can benaturally mated with a female wild type pig, thereby obtaining pancreasformation-inhibited newborns always at a certain ratio.

The newborn piglet in which pancreas formation is inhibited to be usedin this

Example was obtained by natural mating with such a chimeric pig.

Example 2 Raising Newborn Piglet in which Pancreas Formation isInhibited

A newborn piglet in which pancreas formation is inhibited (precisely,that has only rudimentary pancreatic tissue) by carrying a Pdx1-Hes1gene lacks an endocrine cell (for example, a β cell producing insulin)and an exocrine cell. Consequently, the piglet dies about three daysafter birth due to extreme hyperglycemia and dyspepsia.

Insulin and a digestive enzyme combination were administered asappropriate to such a newborn piglet in which pancreas formation isinhibited to bring the blood glucose level close to a normal level andinduce individual growth and development of the pancreas.

1) Insulin Preparation:

Levemir (Novo Nordisk) was administered at a dose of 0.2 to 0.3 IU/kgtwice a day, or Tresiba (Novo Nordisk) was administered once a day.

2) Insulin Administration Method:

Insulin was administered by subcutaneous injection or continuouslyadministered with an insulin pump.

3) Digestive Enzyme Combination Agent:

A daily dose of 150 mg to 750 mg of Berizym (SHIONOGI & CO., LTD.) wasmixed with a milk substitute or solid (powdered) laboratory chow andgiven to the piglet. Berizym contains lipase, cellulase, biodiastase,and pancreatin.

4) Laboratory Chow and Feeding Method:

From 0 to 7 days after birth, the piglet was fed with the milksubstitute by a human or fed with milk by the mother pig. From 8 daysafter birth onward, the piglet was subjected to restricted feeding (thepiglet is not allowed to eat ad libitum and is fed with a fixed dailyamount of food) using a powdered milk substitute or powdered laboratorychow.

Example 3 Development of Pancreas by Supported Raising of Newborn Pigletin which Pancreas Formation is Inhibited

1) Controlled Blood Glucose Level and Growth:

When a newborn piglet in which pancreas formation is inhibited wasraised according to the method described in Example 2, the blood glucoselevel thereof was controlled to some extent and the individual grew asshown in FIG. 2.

2) Blood Biochemical Parameters:

Blood biochemical parameters for the pig in which pancreas formation isinhibited (FIG. 3) indicate that insulin production remained abolishedand glucagon production occured as the pig grew. Glucagon production wasalso confirmed by immunostaining of the developed pancreatic tissue(FIG. 4). In contrast, no insulin production was observed byimmunostaining of the pancreatic tissue, either (FIG. 4).

3) Development of Pancreas:

Supported raising of the newborn piglet in which pancreas formation isinhibited led to development of the rudimentary pancreatic tissue (FIGS.5 and 6). Although the pancreatic tissue was about one tenth as big as awild type pancreatic tissue (FIG. 7), amylase production was alsoobserved (FIG. 8).

Example 4 Transplantation of Autologous Cells into Pancreatic TissueLacking β Cell

The pancreatic tissue developed by supported raising of the newbornpiglet in which pancreas formation is inhibited lacks a β cell, which isa specific functional cell, but has other functional cells and tissue.Therefore, it is presumed that transplanting into this tissue aprogenitor cell of the specific functional cell or a pluripotent stemcell whose differentiation was appropriately induced allows forestablishment and growth of the specific functional cell.

To verify this, autologous cells (fibroblasts) were injected into thepancreatic tissue of a 3.5-month-old pig in which pancreas formation isinhibited. Consequently, the autologous cells formed a colony (FIG. 9).It is presumed that when the cell to be transplanted is a progenitorcell of a β cell or a pluripotent stem cell, β cells can be formed andgrown by utilizing the environment of the pancreatic tissue.Specifically, it is believed that human pancreatic functional cells (forexample, β cells) can be developed by allowing human pluripotent stemcells to establish themselves in the environment within the developingpancreatic tissue of the pig in which pancreas formation is inhibited.

Example 5 Disease of Pig in which Pancreas Formation is Inhibited

1) Diabetic Retinopathy

A newborn piglet in which pancreas formation is inhibited was raisedaccording to the method described in Example 2. Theimmunohistochemically stained image of the retina was observed after 80days of raising and a pathological image characteristic of diabeticretinopathy was identified (FIG. 10). On the other hand, the retina of awild type pig was healthy after raising for the same length of time(FIG. 10).

2) Cataract

A newborn piglet in which pancreas formation is inhibited was raisedaccording to the method described in Example 2. The eye was observedafter 39 days of raising and development of cataract was identified(FIG. 11). On the other hand, the eye of a wild type pig was healthyafter raising for the same length of time (FIG. 11).

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

The present invention relates to a non-human animal that has a secondaryorgan lacking a specific functional cell, and thus, is applicable to thefield of industry that handles such an animal.

1-18. (canceled)
 19. A method for establishment and growth of aprogenitor cell of a specific functional cell or a pluripotent stemcell, or a functional cell derived therefrom, comprising transplantingthe progenitor cell or the pluripotent stem cell into an organ-liketissue, wherein the organ-like tissue is formed by a newborn or a fetusof a genetically modified pig and lacks a functional cell derived fromthe pig.
 20. The method according to claim 19, wherein the progenitorcell of the functional cell or the pluripotent stem cell is ahuman-derived cell.
 21. A material in which a progenitor cell of aspecific functional cell or a pluripotent stem cell, or a functionalcell derived therefrom is established and grown, the material lacking afunctional cell derived from the pig and comprising organ-like tissueformed by a newborn or a fetus of a genetically modified pig, whereinthe progenitor cell or the pluripotent stem cell is transplanted intothe organ-like tissue.
 22. The material according to claim 21, whereinthe progenitor cell of the functional cell or the pluripotent stem cellis a human-derived cell.